臺灣博碩士論文加值系統

多重抗藥性是降低腫瘤化學治癒率的原因之一，過去研究顯示腫瘤細胞的細胞膜幫浦蛋白(P醣蛋白)過表現為最主因，其主要機制是將藥物排出至細胞外，而降低抗癌藥物在細胞內的累積，因而降低腫瘤治癒率。本研究利用分子量為5000克/莫耳的單甲基聚乙二醇和生物可降解的聚己內酯團聯共聚合AB2型親疏水二性單甲基聚乙二醇-雙聚己內酯，其合成步驟將單甲基聚乙二醇與雙氫氧甲基丁酸進行酯化反應，再以酸性陽離子樹脂去除保護基，得到雙末端氫氧基單甲基聚乙二醇，最後產物與己內酯單體進行開環反應，獲得聚己內酯分子量為4500克/莫耳的單甲基聚乙二醇-雙聚己內酯，化學結構經過核磁共振光譜和傅立葉轉換紅外線光譜鑑定，且經熱示差掃描卡量計分析圖譜發現當聚合物中含有雙氫氧甲基丁酸時，熔點因其結晶結構而降低，由62.29 oC降至60.73 oC，核磁共振光譜和凝膠滲透層析儀分析分子量分別為19312克/莫耳和15245克/莫耳。此親疏水二性共聚合物在水溶液中自體形成微胞結構，其臨界微胞濃度為43.7×10-3 毫克/毫升。本研究利用單甲基聚乙二醇-雙聚己內酯包覆抗癌藥物艾黴素，以動態散射粒徑分析儀測得粒徑為21.4奈米的含藥物微胞，而不包覆藥物的微胞粒徑為95.1奈米。微胞與巨噬細胞共培養後測量細胞分泌產生的一氧化氮吸收值為0.413-0.472 吸收值/毫克 蛋白質，而不含微胞的控制組測得的吸收值是0.420吸收值/毫克 蛋白質，此結果顯示微胞能避開巨噬細胞的辨識。以紫外線光譜儀測得微胞對艾黴素的承載量率和包覆率分別為2.01%和22.29 %。搭載艾黴素的微胞在pH 5的酸性醋酸鹽緩衝液中經過48小時後的藥物釋放量為50 %，而在pH 7.4的中性磷酸鹽緩衝液下釋放量為40%。此外，餵食人類野生種(MCF-7/WT)與抗藥性(MCF-7/ADR)乳癌細胞螢光染劑(若丹明123)，並以流式細胞儀分析細胞膜上P醣蛋白作用，結果顯示與抗藥性乳癌細胞比較，野生種乳癌細胞內的若丹明123螢光強度明顯較強，而以西方墨點法測量P醣蛋白過表現，相較於MCF-7/WT，MCF-7/ADR在170千道耳吞的位置，顯示P醣蛋白的表現量。添加搭載藥物的微胞與MCF-7/WT和MCF-7/ADR培養液五天後測得的半致死劑量為0.285 微克/毫升和7.476 微克/毫升，相較於單純添加艾黴素的半致死劑量( MCF-7/WT : 0.036 微克/毫升，MCF-7/ADR : 0.937 微克/毫升)，搭載藥物微胞的半致死劑量約高7.9倍，這是因為當藥物被包覆在微胞內時，藥物釋放速率較緩，所以毒殺細胞數較低，導致半致死劑量較高。以共軛焦雷射顯微鏡觀察含艾黴素微胞與單純艾黴素在MCF-7/WT和MCF-7/ADR細胞內分佈，結果顯示搭載艾黴素微胞絕大部分透過胞飲作用累積在細胞質中，而單純艾黴素可透過擴散作用進入細胞核中。以流式細胞儀定量分析在同一螢光強度下，搭載艾黴素微胞和單純艾黴素累積在抗藥性乳癌細胞中的細胞數為83.16 %和50.91 %，這間接證明搭載艾黴素微胞可避開細胞膜上的幫浦蛋白P醣蛋白而達到逆轉抗藥性的功能。因此本研究發現單甲基聚乙二醇-雙聚己內酯微胞可以抑制MCF-7/ADR細胞抗藥性的特性，使更多藥物能進入細胞內以達到毒殺的效果，預期在未來的體內實驗上，能有效抑制腫瘤的生長速度及延長生物的存活率。

Cancer multidrug resistance is associated with plasma membrane expression of P-glycoprotein which results to a reduction of the intracellular drug concentration after activation of P-glycoprotein efflux. In this study, an AB2 type amphiphilic block copolymer was synthesized from biodegradable methoxy poly (ethylene glycol) (mPEG) and poly (ε-caprolactone) (PCL) to encapsulate the antitumor drug, Doxorubicin (DOX). The initiator mPEG was first esterified with 2,2-bis (hydroxymethyl) butyric acid. Removal of the protective group by acidic resin afforded a copolymer with two hydroxyl chain terminals which was subsequently used in a ring-opening polymerization of ε-caprolactone. The AB2 copolymer mPEG-(PCL)2 was characterized by Fourier transform infrared spectroscopy and 1H nuclear magnetic resonance spectroscopy. By increasing the 2,2-bis (hydroxymethyl) butyric acid content in the copolymer, the melting point can decrease from 62.29 oC to 60.73 oC. The molecular weight of the copolymer was calculated as 15,245 g/mol by gel permeation chromatography and 19,312 g/mol by 1H nuclear magnetic resonance. The amphiphilic block copolymer self-assembled into micelles and the critical micelle concentration was 43.7×10-3 mg/mL. The particle size of the empty micelle was measured by dynamic light scattering and found to be 95.1 nm. After loading with doxorubicin, the particle size was 21.4 nm. The drug encapsulation was measured at 22.29% by UV-vis spectrophotometer. The in vitro release study indicated that 50% of the drug was released from micelles at pH 5 acetate buffer and 40 % at pH 7.4 phosphate buffer in the duration of 48 hours. Incubation of macrophage cells with the empty micelles resulted in 0.420 O.D./mg protein and 0.413-0.472 O.D./mg protein NO production for the control and micelle, respectively. This indicated that the micelles could avoid recognition by macrophage cells. Rhodamine 123 assay by flow cytometry and western blot were used to monitor the relative P-glycoprotein expression in human breast cancer cell lines MCF-7/WT and MCF-7/ADR. The results showed that the fluorescence of rhodamine 123 was stronger in MCF-7/WT than MCF-7/ADR. Western blot analysis detected a single band for P-glycoprotein at 170 kDa. The IC50 value of drug-loaded micelle for MCF-7/WT and MCF-7/ADR were 0.285 μg/mL and 7.476 μg/mL, respectively. These values are 7.9-fold higher than the IC50 of free drug for MCF-7/WT and MCF-7/ADR at 0.036 μg/mL and 0.937 μg/mL, respectively. This can be due to the slow release rate of DOX from the micelles. The cell uptake study by confocal laser scanning microscopy (CLSM) showed that drug-loaded micelles accumulated mostly in the cytoplasm instead of the nuclei through endocytosis. In contrast, free drug diffused throughout the cell. In addition the number of cell uptake under the same fluorescence intensity was measured to be 83.16 % and 50.91 % for drug-loaded micelles and free drug in MCF-7/ADR cells. In conclusion, the AB2 copolymer was able to overcome multidrug resistance of breast cancer cells as it can accumulate more in MCF-7/ADR cells compared to free drug. Future in vivo studies could focus on how the drug delivery system can inhibit the tumor growth and prolong the survival rate.

摘要.............................I
ABSTACT.........................III
英文縮寫對照表....................V
誌謝.............................VI
目錄.............................VII
圖目錄...........................X
表目錄...........................XIII
第一章 緒論......................1
1.1研究背景........................1
1.2文獻回顧與探討...................2
1.2.1腫瘤抗藥性的化學治療概念........2
1.2.2以高分子材料為基礎的化學治療.....4
1.3研究動機與目的...................13
第二章 材料與實驗方法.................15
2.1實驗架構..........................15
2.2實驗用儀器.......................16
2.3實驗用藥品........................18
2.4藥物載體高分子的合成步驟.............22
2.4.1本實驗的化學反應式.................22
2.4.2 α-methoxy-ω-N,N-bis(hydroxyehtyl) poly(ethylene glycol) 合成...................23
2.4.3 Acetonide-2,2-bis(hydroxymethyl)butyric acid合成與鑑定............................23
2.4.4 Acetonide mPEG-BHB合成與鑑定........24
2.4.5移除acetonide protective group......24
2.4.6 mPEG-(PCL)2 合成與鑑定..............25
2.4.7 測量高分子形成微胞的濃度..............25
2.4.8 計算臨界微胞濃度.....................26
2.5 製備包覆艾黴素的微胞....................26
2.5.1 製備不含艾黴素微胞....................26
2.5.2 製備搭載艾黴素的微胞..................26
2.5.3 搭載艾黴素微胞的藥物釋放曲線...........27
2.6 搭載艾黴素微胞對乳癌細胞的體外實驗........28
2.6.1實驗藥品配置...........................28
2.6.2乳癌細胞株之培養........................29
2.6.3檢測乳癌細胞的doubling time.............31
2.6.4生物相容性檢測巨噬細胞NO assay...........32
2.6.5 Rhodamine 123分析野生種和抗藥性乳癌細胞的P-glycoprotein幫浦效應........................32
2.6.6 西方墨點法分析野生種和抗藥性乳癌細胞P-glycoprotein的表現..........................33
2.6.7 搭載艾黴素微胞對乳癌細胞的毒殺作用........37
2.6.8 觀察乳癌細胞攝入搭載艾黴素微胞與單純艾黴素的差異..........................................38
2.6.9統計分析方法 ............................38
第三章 結果與討論.............................39
3.1 藥物載體高分子的合成......................39
3.2 MPEG-(PCL)2微胞與搭載艾黴素微胞的特性與藥物釋放分析..........................................42
3.3搭載艾黴素微胞對乳癌細胞的體外實驗............44
3.3.1乳癌細胞的doubling time..................44
3.3.2生物相容性檢測巨噬細胞NO assay.............46
3.3.3西方墨點法分析抗藥性乳癌細胞表現的P-glycoprotein.................................47
3.3.4 Rhodamine123分析抗藥性乳癌細胞的P-glycoprotein幫浦的效應....................................48
3.3.5搭載艾黴素微胞與單純艾黴素對乳癌細胞的毒殺作用...........................................49
3.3.6觀察乳癌細胞攝入搭載艾黴素微胞與單純艾黴素的差異...........................................50
第四章 結論...................................52
圖...........................................54
表...........................................86
參考文獻......................................87


圖目錄
圖1-1細胞膜上P-glycoprotein幫浦作用機制……………3
圖1-2高分子聚合微胞……………………………………5
圖1-3樹枝狀聚合物..............................6
圖1-4微脂體……………………………………………………8
圖1-5 Enhance permeability and retention 效應………………………………………………………………10
圖1-6單純DAF和含DAF的TMRCA-PCL-b-PEO微胞在細胞內之途徑…………………………………………………………………12
圖2-1藥物載體高分子的合成步驟……………………………22
圖3-1 BHB的1H-NMR圖譜…………………………………54
圖3-2 Acetonide-BHB的1H-NMR圖譜……………55
圖3-3 Acetonide mPEG-BHB的1H-NMR圖譜……………56
圖3-4 mPEG-BHB的1H-NMR圖譜……………………………57
圖3-5 mPEG-(PCL)2的1H-NMR圖譜………………………58
圖3-6 mPEG-BHB的FT-IR圖譜……………………………59
圖3-7 mPEG-BHB與mPEG-OH的FT-IR圖譜比較..............................................60
圖3-8 mPEG-(PCL)2的FT-IR圖譜…………61
圖3-9 mPEG-BHB與mPEG-OH的DSC第一次升溫曲線比較圖………………………………………………………………62
圖3-10 mPEG-OH、mPEG-BHB與mPEG-(PCL)2的GPC圖譜...............................63
圖3-11 mPEG-(PCL)2的臨界微胞濃度曲線圖......................................64
圖3-12 mPEG-(PCL)2 微胞的粒徑大小分佈圖.............................................65
圖3-13 DOX藥物濃度的標準曲線………………………………………………66
圖3-14 mPEG-(PCL)2 搭載DOX微胞的粒徑大小分佈圖………………………67
圖3-15 mPEG-(PCL)2 搭載DOX微胞在pH 7.4和pH 5環境下的藥物釋放曲線圖…………………………….68
圖3-16 MCF-7/WT和MCF-7/ADR細胞的生長曲線圖……69
圖3-17不同濃度的mPEG-(PCL)2 微胞與LPS對巨噬細胞產生的細胞型態變化..............................70
圖3-18 不同濃度的mPEG-(PCL)2 微胞對巨噬細胞產生NO分析......................71
圖3-19西方墨點法分析MCF-7/WT 與MCF-7/ADR細胞膜上P-glycoprotein的表現………………………………………………………72
圖3-20螢光顯微鏡觀察rhodamine123於MCF-7/WT和MCF-7/ADR乳癌細胞內的分布…………………………………………………………………73
圖3-21流式細胞儀分析MCF-7/ WT與MCF-7/ADR將rhodamine123攝入細胞內的螢光強度比較………………………………………………………………….74
圖3-22 mPEG-(PCL)2搭載DOX微胞和單純DOX對乳癌細胞的IC50分析…………………………………………………………75
圖3-23 MCF-7/ WT將單純DOX攝入細胞內24小時後的螢光分佈之共軛焦顯微鏡圖譜…………………………………………………………………76
圖3-24 MCF-7/ WT將mPEG-(PCL)2 搭載DOX微胞攝入細胞內24小時後的螢光分佈之共軛焦顯微鏡圖譜……………………………………………………….77
圖3-25 MCF-7/ ADR將單純藥物攝入細胞內24小時後的螢光分佈之共軛焦顯微鏡圖譜……………………………………………………………………78
圖3-26 MCF-7/ ADR將mPEG-(PCL)2 搭載DOX微胞攝入細胞內24小時後的螢光分佈之共軛焦顯微鏡圖譜……………………………………………………….79
圖3-27流式細胞儀分析MCF-7/ WT將mPEG-(PCL)2 搭載DOX微胞與單純DOX攝入細胞內的螢光強度差異……………………80
圖3-28 流式細胞儀分析MCF-7/ ADR將mPEG-(PCL)2 搭載DOX微胞與單純DOX攝入細胞內的螢光強度差……………………………………………………81
圖3-29 MCF-7/ WT將單純DOX攝入細胞內24小時後的螢光分佈之共軛焦顯微鏡xy軸系列圖譜………………………………………………………………82
圖3-30 MCF-7/ WT將mPEG-(PCL)2 搭載DOX微胞攝入細胞內24小時後的螢光分佈之共軛焦顯微鏡xy軸系列圖譜……………………………………………83
圖3-31 MCF-7/ ADR將單純DOX攝入細胞內24小時後的螢光分佈之共軛焦顯微鏡xy軸系列圖譜………………………………………………………………....84
圖3-32 MCF-7/ ADR將mPEG-(PCL)2搭載DOX微胞攝入細胞內24小時後的螢光分佈之共軛焦顯微鏡xy軸系列圖譜……………………………………………85


表目錄
表2-1儀器設備目錄…….…….……………………16
表2-2藥品目錄……………..………………18
表3-1 mPEG5000-(PCL4500)2 共聚合物特性……………………………………..… .86

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